Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE

Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by sonication, cell supernatants are separated using Tris- Glycine SDS-PAGE, and superfolder GFP (sfGFP) fluorescence is directly visualized within gels. This method is rapid, with sfGFP immediately imaged following SDS-PAGE without staining. Further, sfGFP can be specifically detected in complex samples such as E. coli cell supernatants, proteins run at expected masses, and all steps can be performed at ambient temperature. This strategy is broadly applicable beyond the study of protein splicing and can be used for sensitive and specific visualization of superfolder sfGFP-tagged proteins in-gel

Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing

Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-target Considerations

Protein splicing is a post-translational process by which an intervening polypeptide, or intein, catalyzes its own removal from the flanking polypeptides, or exteins, concomitant with extein ligation. Although inteins are highly abundant in the microbial world, including within several human pathogens, they are absent in the genomes of metazoans. As protein splicing is required to permit function of essential proteins within pathogens, inteins represent attractive antimicrobial targets. Here we review key proteins interrupted by inteins in pathogenic mycobacteria and fungi, exciting discoveries that provide proof of concept that intein activity can be inhibited and that this inhibition has an effect on the host organism’s fitness, and bioanalytical methods that have been used to screen for intein activity. We also consider potential off-target inhibition of hedgehog signaling, given the similarity in structure and function of inteins and hedgehog autoprocessing domains

A Radial Velocity Survey of the Cygnus OB2 Association

We conducted a radial velocity survey of the Cygnus OB2 Association over a 6 year (1999 - 2005) time interval to search for massive close binaries. During this time we obtained 1139 spectra on 146 OB stars to measure mean systemic radial velocities and radial velocity variations. We spectroscopically identify 73 new OB stars for the first time, the majority of which are likely to be Association members. Spectroscopic evidence is also presented for a B3Iae classification and temperature class variation (B3 - B8) on the order of 1 year for Cygnus OB2 No. 12. Calculations of the intial mass function with the current spectroscopic sample yield Gamma = -2.2 +/- 0.1. Of the 120 stars with the most reliable data, 36 are probable and 9 are possible single-lined spectroscopic binaries. We also identify 3 new and 8 candidate double-lined spectroscopic binaries. These data imply a lower limit on the massive binary fraction of 30% - 42%. The calculated velocity dispersion for Cygnus OB2 is 2.44 +/- km/s, which is typical of open clusters. No runaway OB stars were found.Comment: 56 pages, 23 figures, 5 tables, accepted for publication in the Astrophysical Journa

Massive stars in extremely metal-poor galaxies: a window into the past

Cosmic history has witnessed the lives and deaths of multiple generations of massive stars, all of them invigorating their host galaxies with ionizing photons, kinetic energy, fresh material, and stellar-mass black holes. Ubiquitous engines as they are, astrophysics needs a good understanding of their formation, evolution, properties and yields throughout the history of the Universe, and with decreasing metal content mimicking the environment at the earliest epochs. Ultimately, a physical model that could be extrapolated to zero metallicity would enable tackling long-standing questions such as “What did the first, very massive stars of the Universe look like?” or “What was their role in the re-ionization of the Universe?” Yet, most of our knowledge of metal-poor massive stars is drawn from one single point in metallicity. Massive stars in the Small Magellanic Cloud (SMC, ∼1/5Z⊙ ) currently serve as templates for low-metallicity objects in the early Universe, even though significant differences with respect to massive stars with poorer metal content have been reported. This White Paper summarizes the current knowledge on extremely (sub-SMC) metal poor massive stars, highlighting the most outstanding open questions and the need to supersede the SMC as standard. A new paradigm can be built from nearby extremely metal-poor galaxies that make a new metallicity ladder, but massive stars in these galaxies are out of reach to current observational facilities. Such a task would require an L-size mission, consisting of a 10m-class space telescope operating in the optical and the ultraviolet ranges. Alternatively, we propose that ESA unites efforts with NASA to make the LUVOIR mission concept a reality, thus continuing the successful partnership that made the Hubble Space Telescope one of the greatest observatories of all time

An Atlas of FUSE Sight Lines Toward the Magellanic Clouds

We present an atlas of 57 Large Magellanic Cloud (LMC) and 37 Small Magellanic Cloud (SMC) observations obtained with the Far Ultraviolet Spectroscopic Explorer (FUSE) satellite. The atlas highlights twelve interstellar absorption line transitions at a resolution of ~15 km/s. These transitions cover a broad range of temperatures, ionization states, and abundances. The species included are OVI, which probes hot (T~3x10^5 K) ionized gas; CIII and FeIII, which probe warm (T~10^4 K) ionized gas; SiII, PII, CII, FeII, and OI, warm neutral gas; and six different molecular hydrogen transitions, which trace cold (T<=500 K) gas. We include Schmidt Halpha CCD images of the region surrounding each sight line showing the morphology of warm ionized gas in the vicinity, along with continuum images near each FUSE aperture position. Finally, we present several initial scientific results derived from this dataset on the interstellar medium of the Magellanic Clouds and Galactic halo.Comment: 29 pages, 6 figures. Complete Atlas of 94 additional images (~800kB each) is available at http://fuse.pha.jhu.edu/~danforth/atlas Accepted to the ApJS March 200

Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.National Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473

The Near-Earth Object Surveyor Mission

The Near-Earth Object (NEO) Surveyor mission is a NASA observatory designed to discover and characterize near-Earth asteroids and comets. The mission's primary objective is to find the majority of objects large enough to cause severe regional impact damage ($>$140 m in effective spherical diameter) within its five-year baseline survey. Operating at the Sun-Earth L1 Lagrange point, the mission will survey to within 45 degrees of the Sun in an effort to find the objects in the most Earth-like orbits. The survey cadence is optimized to provide observational arcs long enough to reliably distinguish near-Earth objects from more distant small bodies that cannot pose an impact hazard. Over the course of its survey, NEO Surveyor will discover $\sim$200,000 - 300,000 new NEOs down to sizes as small as $\sim$10 m and thousands of comets, significantly improving our understanding of the probability of an Earth impact over the next century.Comment: accepted to PS

Minimal Mesoscale Model for Protein-Mediated Vesiculation in Clathrin-Dependent Endocytosis

In eukaryotic cells, the internalization of extracellular cargo via the endocytic machinery is an important regulatory process required for many essential cellular functions. The role of cooperative protein-protein and protein-membrane interactions in the ubiquitous endocytic pathway in mammalian cells, namely the clathrin-dependent endocytosis, remains unresolved. We employ the Helfrich membrane Hamiltonian together with surface evolution methodology to address how the shapes and energetics of vesicular-bud formation in a planar membrane are stabilized by presence of the clathrin-coat assembly. Our results identify a unique dual role for the tubulating protein epsin: multiple epsins localized spatially and orientationally collectively play the role of a curvature inducing capsid; in addition, epsin serves the role of an adapter in binding the clathrin coat to the membrane. Our results also suggest an important role for the clathrin lattice, namely in the spatial- and orientational-templating of epsins. We suggest that there exists a critical size of the coat above which a vesicular bud with a constricted neck resembling a mature vesicle is stabilized. Based on the observed strong dependence of the vesicle diameter on the bending rigidity, we suggest that the variability in bending stiffness due to variations in membrane composition with cell type can explain the experimentally observed variability on the size of clathrin-coated vesicles, which typically range 50–100 nm. Our model also provides estimates for the number of epsins involved in stabilizing a coated vesicle, and without any direct fitting reproduces the experimentally observed shapes of vesicular intermediates as well as their probability distributions quantitatively, in wildtype as well as CLAP IgG injected neuronal cell experiments. We have presented a minimal mesoscale model which quantitatively explains several experimental observations on the process of vesicle nucleation induced by the clathrin-coated assembly prior to vesicle scission in clathrin dependent endocytosis

Current practice and surgical outcomes of neoadjuvant chemotherapy for early breast cancer : UK NeST study

Funding Information: This work was funded by a grant from the Association of Breast SurgeryPeer reviewedPublisher PD